This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to: (i) continue investigating the post-translational chemistry for the green fluorescent protein (GFP) family and (ii) test algorithm-based design methodology through the construction of metal-binding GFP variants with potential applications as in vivo biosensors. GFP has revolutionized molecular tagging and cell labeling, including applications in protein trafficking, gene expression, and the study of pathogen function and human disease. GFP (and its homologs) are well suited for high-resolution structural, spectroscopic, mutational, and computational studies that reveal in atomic detail how proteins self-synthesize their chromophores and tune the chromophore?s photophysical properties for chemical and biological function. Initial studies made possible by high resolution SSRL crystallographic data allowed us to identify remarkable and unprecedented spontaneous amino acid modifications in GFP that include oxygen incorporation, peptide bond hydrolysis, oxidative cross links, decarboxylation, and carbon-carbon bond cleavage reactions. Moreover, the ability to reproducibly obtain crystals of variants makes GFP an excellent design target scaffold. The rational design of metalloproteins with desired functional properties has tremendous potential for biotechnological or medial applications. We are using an algorithm-based methodology (DEZYMER) to design metal-binding sites into GFP as a first step towards metalloprotein functional design. In addition, we aim to link the designed metal site to the fluorescent properties of the GFP chromophore to create a novel reporter system that permits monitoring of in vivo metal ion concentrations. Using rounds of recursive design, made possible by high resolution data collected at SSRL, we have created multiple metal site designs that modulate GFP fluorescent properties. High resolution structural analysis of these metal ion biosensors, along with their design intermediates and apo structures, allow us to close the design cycle and rigorously evaluate and